This R21 application proposes to develop the norovirus (NV) P particle into a vaccine platform to fulfill the need of control and prevention of infectious diseases. This NV P particle is formed by 24 copies of the protruding (P) domain of NV capsid which has an ideal size for a subunit vaccine. The P particles can be readily produced in E. coli with simple purification procedures and low cost. The P particles are highly stable and immunogenic. In this study we will take advantage of the 3 surface loops of each P domain for foreign antigens presentation by insertion of the antigens into the surface loops. We have proven this concept by insertion of various small epitopes and large antigens on one of these loops, including the M2e epitope of influenza virus and rotavirus (RV) surface antigen VP8s. The P particle-M2e and -VP8 chimeras induced high titers of neutralizing antibodies against replication of influenza virus and RV and protected vaccinated mice from infection of the two viruses, respectively, in mouse challenge models. In addition, the resulting antibodies blocked NV-receptor attachment. In this study we will improve the P particle vaccine platform for broader applications by systematically assessing the capability and capacity of the other surface loops of the P particle to present foreign antigens and by construction of new user-friendly P particle vectors. In addition, we will perform challenge study using mouse model to improve the immune enhancement and production efficiency of the P particle platform through 1) increase of copy number of an inserted antigen, 2) addition of immunogenic stimulus molecules to the antigens, and 3) addition of a purification tag to the chimera. This study will gather further evidence on the usefulness of the P particle platform for antigen presentation. The proposed research herein that base logically on our prior work combines the strengths of three labs of Drs. Tan, Jiang and McNeal at the Cincinnati Children's Hospital Medical Center with expertises in molecular biology, structural biology, and virology, immunology and vaccine technology. With the large body of promising preliminary data, our track-records on research and the collaborative spirit between our research teams, we are confident that we will fully the proposed research to develop the NV P particle platform for presenting diverse foreign antigens and move the science forward. PUBLIC HEALTH RELEVANCE: In this application we proposed to develop our recently discovered subviral particle, the norovirus (NV) P particle, into a vaccine platform for antigen presentation and thus for novel vaccine development. The P particle is composed of 24 copies of NV protruding (P) domains. Each P domain in the P particle has three exposed loops on its distal surface and these loop have been shown to be excellent sites for foreign antigen presentation. Antigens can be inserted onto one to three surface loops of the P domain by molecular cloning. The resulting recombinant protein will spontaneously form chimeric P particle that contain at least 24 copies of the inserted antigen on the surface of the P particle. The P particles can be easily produced in E. coli, are highly stable and highly immunogenic and, therefore, are an ideal vaccine platform. We have accumulated a large amount of data in insertion and presentation of several protein and peptide antigens on the P particle, including the M2e epitope of influenza virus and the surface antigen VP8 of rotavirus (RV). In this application we will gather further evidence on usefulness of P particle as a platform for vaccine development against various infectious diseases. Two lines of experiments will be performed to fulfill our goals. In Aim 1 the capability and capacity of individual surface loops for small-to-large antigen insertions will be systematically assessed for a maximal application of the P particle platform. To facilitate these experiments, user-friendly P particle vectors with a convenient cloning cassette in each loop will be generated. Our goal is to maximize the usefulness of the P particle vaccine platform. In Aim 2 the usefulness of the P particle vaccine platform will be further evaluated by mouse challenge studies on a human flu virus and a mouse RV. Particular attention will be paid to the improvement of immune enhancement by increasing the copy number of an antigen per P particle. This will be achieved through an insertion of a repeated M2e epitopes in individual loops of the P particle. We also will examine whether further immune enhancement can be reached by an extra antigenic stimulus molecule to be inserted to the P particles-M2e and -VP8 chimeras. Through these studies we wish to further build up confidence on our research which may help to speed up the process of commercialization of the P particle vaccine platform for a broad application by many laboratories around world.